Proposal of International Gluten Research Group
In a scenario of climate change and rapidly rising urban populations demanding processed foods, it is necessary to develop new wheat cultivars combining high yield potential, disease resistance, and stability for yield and processing quality, even under heat or drought stress conditions. Allelic variation for gluten proteins (glutenin subunits and gliadins) is a major determinant of differences in dough viscoelastic properties observed between cultivars of both bread wheat and durum wheat. Technical difficulties in allelic identification due to the complexity of the protein profile produced by each cultivar and the use of different nomenclature systems in different laboratories has historically interfered with information exchange between research groups, a situation exacerbated by the vast number of possible profiles found in different cultivars due to the multi-allelic nature of the principal loci encoding gluten proteins (Glu-1, Glu-2, Glu-3, Gli-1 and Gli-2). For the Glu-3 alleles, we have collaborated to unify criteria across laboratories and to compare four different methods of allelic identification (SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR), and have shown that the four methods can be regarded as complementary techniques for allelic identification. We seek to continue addressing remaining analytical challenges, place the findings in the context of the Catalogue of Gene Symbols for Wheat, and, with unified criteria, initiate work to define better the relationship between specific gluten proteins and processing quality attributes. Therefore, we propose a new system to share materials through public gene banks in collaboration with the Catalogue, and the formation of a wider international group aimed at facilitating the resolution of the remaining problems in the field. We also propose to extend our collaboration by forming a wheat quality expert working group under the Wheat Initiative.
KeywordsDurum Wheat Common Wheat Gluten Protein Glutenin Subunit Wheat Quality
Current Status of Glu-3 Allele Nomenclature
It has been shown that allelic variation for the high-molecular-weight glutenin subunits (HMW-GSs) and low-molecular-weight glutenin subunits (LMW-GSs) affects the properties of dough made with different wheat cultivars. LMW-GS composition in common wheat is one of the critical determinants of gluten properties (Branlard et al. 2001; Eagles et al. 2006; Gupta et al. 1994; Liu et al. 2005; Maruyama-Funatsuki et al. 2005). Gupta and Shepherd (1990) assigned the individual LMW-GSs to Glu-A3, Glu-B3 and Glu-D3 loci and selected standard cultivars that covered the allelic variation observed. However, subsequent use of Glu-3 nomenclature has not been consistent among laboratories, due to the complexity of the LMW-GSs, different separation methods and different standard cultivars used by researchers (Branlard et al. 2003; Ikeda et al. 2006; Singh et al. 1990). It is necessary to unify the various Glu-3 allelic nomenclature systems in use, to allow information to be shared regarding the effects of individual alleles on gluten properties and to be applied in breeding programs aimed at improving gluten properties. In previous studies, four laboratories plus an international institution shared cultivars and compared results. We confirmed that there were inconsistencies to identify Glu-3 alleles between laboratories due to differences of separation and identification methods (Ikeda et al. 2008). Using 2-DE analysis, we found new Glu-3 alleles among these materials (Ikeda et al. 2009). Combining SDS-PAGE, 2-DE, MALDI-TOF-MS and PCR analyses, we showed that a combination of methods was required to identify certain alleles, and that these methods would be especially useful when characterizing new alleles. We also recommended 30 cultivars as standards for the determination of LMW-GS alleles (Liu et al. 2010).
It is also important to use common methods to identify Glu-3 alleles. For SDS-PAGE, Peña proposed the use of separation gels containing Tris buffer of pH 8.5 instead of pH 8.8 for better separation of LMW-GS bands (Ikeda et al. 2008; Peña et al. 2004). Lowering bis-acrylamide concentration and using larger size gels also helps better separation (Branlard et al. 2003). Further evaluation for creating a standard SDS-PAGE method is necessary. For PCR markers, as the number of known alleles increases, we need to reconfirm the usefulness of PCR markers to identify Glu-3 alleles. For example, a PCR primer set, which was developed to identify the Glu-B3i allele (Wang et al. 2009), identified the Glu-B3ad allele instead. It is necessary to select a standard PCR primer set to identify Glu-3 alleles.
Functional Analysis of Gluten Proteins
By sharing materials and methods among international research groups, it becomes possible to define better the relationship between specific gluten proteins and processing quality stability, even under heat or drought stress wheat growing conditions. We will set an international framework to evaluate allelic effects on quality attributes under various environmental conditions.
Unification with Durum Glu-3 Alleles
Durum Glu-3 alleles were classified independently of those of common wheat (Martinez et al. 2004; Nieto-Taladriz et al. 1997). In the Catalogue, the durum Glu-3 alleles were originally assigned separately and subsequently combined into one provisional list. Since tetraploid durum wheat shares common ancestral species with common wheat, we would expect some alleles to be identical to those of common wheat. We shared standard cultivars and studied Glu-3 alleles by SDS-PAGE, 2-DE and PCR. Some alleles seemed to share the same alleles with common wheat, but some were unique in durum wheat (data not shown). This means that durum allele might widen the genetic diversity of common wheat alleles, and vice versa. Further analysis is necessary to clarify durum Glu-3 alleles and produce a definitive list in the Catalogue for use by the wheat community. This is also important for Glu-1 alleles.
Gliadin consists of α/β/γ/ω-gliadins, which contain many proteins having a range of molecular weights and pI values. Variation in the gliadins also effects dough properties (Branlard and Dardevet 1994). Gliadins are also known to contain epitopes involved in wheat gluten related disorders (Sapone et al. 2012). Gliadin analysis was mainly carried out using A-PAGE. The analysis of gliadin proteins using SDS-PAGE allows the determination of the banding patterns associated with the close linkage existing between Gli-1 and Glu-3, and, therefore, this approach further contributes to the identification of specific Glu-3 LMW-GS in both common and durum wheat. With increasing genome sequence data availability, it is important to identify gliadins by proteomic techniques to clarify correspondence between gliadin proteins, the epitopes of allergen and coding genes (Juhasz et al. 2012).
Forming an International Gluten Research Group
The field of gluten research overlaps other wheat quality related fields, e.g. allergy, nutrition and carbohydrates. Therefore, it is logical to attempt extending our collaboration to other researchers related to wheat quality. Currently we work to form a wheat quality expert working group under the Wheat Initiative (http://www.wheatinitiative.org/about/expert-working-groups). We would like to invite other colleagues related to wheat quality to join our collaboration.
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